ndividuals with either types I or II diabetes are 2- to 4- times more likely to develop cardiovascular disease (CVD) than 1on-diabetics. This risk is particularly high among those with hyperglycemia, and probably insulin resistance, and it ,_scapes quantification based upon traditional CVD risk factors alone. One emerging feature of diabetes is an excess of oxidative stress that, in the vasculature, has a number of important sequelae including altered NO bioactivity, increase( adhesion molecule expression, and the promotion of atherosclerotic lesion formation. The precise mechanism(s) whereby oxidative stress promotes these phenotypic characteristics of vascular disease are not known. In preliminary data presented here, we have found that two hallmarks of types I and II diabetes, hyperglycemia and elevated free fatt3 acids (FFAs), impart an oxidative stress in cultured endothelial cells that mediates several maladaptive responses including insulin resistance, eNOS-mediated oxidant production, and reduced NO bioactivity. We have also demonstrated AMP kinase activation in response to oxidative stress that we view as a central for the cellular adaptatiol to oxidative stress The goal of this proposal is to determine the role of AMP kinase activation in the phenotypic _bnormalities of the endothelium promoted by oxidative stress due to excess glucose and FFAs. To accomplish this oal, we will first characterize the nature of the oxidative stress due to hyperglycemia/FFAs with particular attention tt the mitochondrion, NADPH oxidase, and eNOS as sources of this oxidative stress. We will then work in close collaboration with Project 1 to determine if oxidative stress is a cause or consequence of changes in cellular metabolism due to hyperglycemia/FFAs. We will then be in a position to determine how cellular metabolism and oxidative stress from hyperglycemia/FFAs relate to endothelial cell insulin resistance and Akt signaling. Since AMP kinase is a key factor in cellular adaptation to stress, we will then characterize the activation of AMP kinase by hyperglycemia/FFAs and the role of oxidative stress in this process. The molecular consequences of AMP kinase activation will also be determined in order to gain insight into the mechanisms of reduced oxidative stress by AMP kinase activation. Finalb we will determine the consequences of hyperglycemia/FFAs for endothelial cell NO bioactivity with particular attention to eNOS phosphorylation status and the resultant consequences for catalytic activity and eNOS-mediated oxidant production. Using this approach, we should be able to define the pathologic events associated with hyperglycemia an, FFAs that contribute to diabetic CVD.